ABSTRACT Graft-versus-host disease (GVHD) is a leading cause of non-relapse mortality after allogeneic hematopoietic cell transplantation (alloHCT). Current GVHD prophylaxis relies on broadly suppressive tacrolimus-based combinations that fail to tolerize donor T-cells. Tacrolimus inhibits T-cell receptor (TCR) activation and impairs regulatory T-cell (Treg) longevity and function. Rather than targeting the TCR, the novel approach tested in this application is concurrent blockade of T-cell costimulation and cytokine activation as a strategy to spare Tregs, prevent GVHD, and maintain graft-versus-leukemia (GVL). CD28 costimulation of T-cells requires signal transduction by mTOR and Aurora kinase, where inhibiting either molecule ameliorates GVHD in rodents. The IL-6 receptor directs JAK2 to phosphorylate STAT3, which polarizes pathogenic Th1 and Th17 development over beneficial Tregs. We observed that STAT3 activity is increased in alloHCT recipients who later develop acute GVHD. Blockade of JAK2 or STAT3 also reduces murine GVHD. Our data support the concept that dual inhibition of CD28 and IL-6 activity is synergistic and offers durable GVHD prevention with intact GVL. Aim 1 of this application will test this hypothesis in a proof-of-principle clinical trial by combing pacritinib with sirolimus-based immune suppression to respectively target JAK2 and mTOR signaling and reduce GVHD. Aims 2 and 3 will test the hypotheses that neutralization of downstream signaling molecules directed by CD28 and IL-6 will yield distinct immune outcomes and collectively more complete control over donor T-cells. Unlike JAK2, STAT3 molecularly counteracts Treg development. We provide evidence that JAK2 blockade permits natural Treg development, while STAT3 inhibition increases inducible Tregs (iTreg). In Aim 2, we will investigate whether STAT3/mTOR inhibition is better than JAK2/mTOR inhibition in reducing GVHD in mice by enhancing iTreg differentiation. We provide evidence that inhibition of Aurora kinase significantly increases iTreg suppressive potency. In Aim 3, we will determine whether targeting STAT3/Aurora is better than JAK2/Aurora to prevent GVHD by increasing both iTreg differentiation and function. These experiments will direct future translation and development of tacrolimus-free GVHD prophylaxis regimens. Our long-term goal is to develop selective immune suppression strategies that effectively prevent GVHD and maintain essential T-cell responses needed to preserve GVL.